Many efforts have recently been made to improve growth and resistance of crop plants. Some of the most important crop plants, e.g. rice, wheat, barley, potato, belong to the so-called C3 plants. Only a few important crop plants, like corn and sugar cane, are C4 plants. CO2 fixation in C3 plants is primarily catalyzed by the enzyme ribulose-1,5-bisphosphate carboxylase (RUBISCO) which is located in the chloroplasts. The enzyme RUBISCO catalyzes two reactions: carboxylation and oxygenation of ribulose-1,5-bisphosphate. The product of the first reaction are two molecules of 3-phosphoglycerate which enter the Calvin cycle to form starch and ribulose-1,5-bisphosphate. The products of the oxygenase reaction are one molecule each of 3-phosphoglycerate and phosphoglycolate (Goodwin and Mercer, 1983). The latter is converted to 3-phosphoglycerate in a biosynthetic pathway known as photorespiration (see FIG. 1). In the course of this complex sequence of reactions, one molecule of CO2 is released and lost for the plant. This loss of CO2 reduces the formation of sugars and polysaccharides in the plant and thus reduces their productivity. Furthermore, NH3 is released which has to be refixed. These effects are exacerbated further when plants are grown under suboptimal water supply. Here, leaf stomata are closed and the intercellular oxygen concentration rises because of molecular oxygen released from the light reactions of photosynthesis. High amounts of phosphoglycolate are produced that enter the photorespiratory cycle. It has been estimated that plants lose approximately 25% of the already fixed carbon due to photorespiration. This cycle is absolutely intrinsic to all C3 plants because of the oxygenase activity of RUBISCO (Leegood et al., 1995; Tolbert, 1997).
The importance of photorespiration for plant growth and yield has been shown by several experiments where the atmospheric CO2 concentration has been artificially raised in greenhouse experiments. Significant increases in the performance of several crop species have been observed already when the CO2 concentration is doubled (Kimball, 1983; Arp et al., 1998). However, this approach is not applicable to the large, open areas used for agricultural production.
C4 plants have evolved a mechanism to avoid these losses. They have employed enzymes already present in their C3 ancestors, but changed the degree of expression as well as the localisation on a subcellular and cell-type specific level. By separating primary and secondary carbon fixation in two different tissues, they drastically increase the local CO2 concentration at the site of RUBISCO activity. Shortly, the first CO2 fixation takes place in the cytoplasm of mesophyll cells and is catalyzed by PEPC, an enzyme without intrinsic oxygenase activity and significantly higher affinity to its substrate compared to RUBISCO. The resulting C4 acid diffuses into the gas tight bundle sheath and is here decarboxylated to liberate CO2. The remaining monocarbonic acid serves to regenerate the primary CO2 acceptor in the mesophyll. This CO2 concentration mechanism results in a complete suppression of photorespiration and an oxygen-insensitive photosynthesis (Kanai and Edwards, 1999). A similar mechanism with a temporal, instead of spatial, separation of enzymatic activities is applied by the crassulacean acid metabolism (CAM) plants (Cushman and Bohnert, 1999).
Beside these mechanisms depending on the cooperation of two different cell types some aquatic plants have developed C4-like mechanisms working within one cell. Here, primary and secondary CO2 fixation take place in one cell, but in different compartments. Whereas PEPC activity is restricted to the cytoplasm, CO2 release and refixation similar to C4 plants take place in the chloroplast. This unicellular C4-like pathway results in a partial suppression of photorespiration with reduced sensitivity to oxygen (Reiskind et al., 1997).
Several attempts have been described to transfer C4- or C4-like pathways or components of this pathway to C3 plants. Mostly, overexpression of PEPC has been used so far. Three groups applied expression of the maize PEPC cDNA or gene under control of different promoters (Hudspeth et al., 1992; Kogami et al., 1994; Ku et al., 1999). Although increases in PEPC activity levels up to 100-fold were detected with the complete intact maize gene in rice (Ku et al., 1999), there were only weak impacts on plant physiology and growth performance (Matsuoka et al., 2001, see also EP-A 0 874 056). Recently, the overexpression of PEPC and malate dehydrogenase from Sorghum in potato has been described, but in this case expression levels were low and no modification of photosynthetic parameters could be observed (Beaujean et al., 2001). PEPC cDNAs from bacterial source have been overexpressed in potato by Gehlen et al. (1996) with some minor impact on photosynthetic parameters. The combination of this enzyme with the additional overexpression of a NADP+-malic enzyme (ME) from Flaveria pringlei targeted to the chloroplast enhanced these effects without any impact on plant growth or yield (Lipka et al., 1999). For rice, it has been recently described that the overexpression of a phosphoenolpyruvate carboxykinase (PCK) from Urochloa panicoides targeted to the chloroplast results in the induction of endogenous PEPC and the establishment of a C4-like cycle within a single cell. However, no enhanced growth parameters were observed (Suzuki et al., 2000; see also WO 98/35030).
Therefore, despite several attempts to improve CO2 fixation and reduce photorespiration, until now no method has been provided that leads to an improvement of growth, productivity, and/or yield for agricultural crop plants. All these attempts were aiming to concentrate CO2 at the site of fixation in order to suppress the oxygenase activity of RUBISCO.
Many bacteria have evolved biochemical pathways to metabolize glycolate, the primary product of the oxygenase activity of RUBISCO. For Escherichia coli, this pathway has been described in great detail (Lord, 1972; Pellicer et al., 1996). E. coli is capable of growing on glycolate as the sole carbon source. As summarised in FIG. 4, glycolate is first oxidized to glyoxylate. This reaction is brought about by a multiprotein complex that is capable of oxidizing glycolate in an oxygen-independent manner. The proteins necessary for glycolate oxidation in E. coli have been analysed and it has been shown that the open reading frames D, E, and F of the glycolate oxidase operon glc are encoding the components of the active enzyme. In the next reaction step, two molecules of glyoxylate are ligated by glyoxylate carboligase (GCL) to form tartronic semialdehyde (TS) and CO2 is released in this reaction. TS is further converted to glycerate by TS reductase (TSR). Glycerate is integrated into the bacterial basal carbon metabolism.
A similar pathway is also applied as a photorespiratory cycle in some green algae and cyanobacteria (Nelson and Tolbert, 1970; Ramazanov and Cardenas, 1992). In this case, glycolate oxidation is seemingly catalysed by glycolate dehydrogenase located inside the mitochondria. Again, this enzyme is not oxygen-dependent and uses organic electron acceptors like Nicotin-Adenosin-Dinucleotid (NAD+) instead. The further metabolism of glyoxylate seems to be similar to the pathway as described for E. coli. 